Background
Cellular compartmentalization is essential for the regulation of metabolism and gene expression (Harrington, Feliu, Wiuf, & Stumpf, 2013). Reciprocal communication between the mitochondria, chloroplasts and nuclei is not only vital for the efficient functions of these compartments, but it also ensures the rapid adjustment of their protein content and composition to changing environmental conditions. Mitochondria-derived and plastid-derived retrograde signals are therefore important components in the regulation of nuclear gene expression (Diaz et al., 2018; Grubler et al., 2017; Kindgreen et al., 2012; Pogson, Woo, Forster, & Small, 2008). Retrograde signalling between the chloroplast and nucleus are not easily distinguished from those that operate during the proplastid-to-chloroplast transition in leaf development because of crossover between the biogenic and operational control of chloroplast functions (Pogson et al., 2008). In plastids, transcription is under the control of two types of RNA polymerases, a unique eubacterial-type plastid-encoded polymerase (PEP) and phage-type nucleus-encoded polymerases (NEPs). These RNA-polymerases specifically regulate the transcription of different subsets of genes but can also co-regulate a portion of the plastidial genes. The formation of chloroplasts from proplastids requires the establishment of the PEP complex. The PEP complex, which is composed of a catalytic core comprised of plastid-encoded proteins (rpoA, rpoB, rpoC1 andrpoC2 ) and additional polymerase-associated proteins (PAP) including other nuclear-encoded polymerase-associated proteins and sigma factors (SIGs), which are required by PEP for promoter recognition( Dietz, & Pfannschmidt, 2011). PEP status/activity provides positive retrograde signals from the chloroplasts that convey essential information to the nucleus to promote PhANG expression.
The WHY family of proteins, which are specific to the plant kingdom (Desveaux et al., 2004) have a putative KGKAAL DNA binding domain that allows binding to ssDNA molecules of differing nucleotide sequence (Grabowski, Miao, Mulisch, & Krupinska, 2008) which may allow them to function as PAPs allowing the possibility of a functional interaction between these proteins (Días et al., 2017).
Mitochondria to nucleus signalling, which involves two key transcription factors: ANAC013 and ANAC017 , is also linked to plastid to nucleus signalling (Shapiguzov et al., 2019). The ANAC013 andANAC017 transcription factors are released from the endoplasmic reticulum upon perception of appropriate signals and translocated to the nucleus, where they activate the expression of a specific set of genes called mitochondrial dysfunction stimulon (MDS) genes that include the alternative oxidases, SOT12 and ANAC013 (De Clercq et al., 2013; Safrany et al., 2008). The enhanced expression of ANAC013provides positive feedback regulation of the signalling pathway. The nuclear-localised RADICAL-INDUCED CELL DEATH1 (RCD1) protein suppresses ANAC013 and ANAC017 functions (Shapiguzov et al., 2019). In addition, SOT12 belongs to the group of MDs genes that overlap with the genes induced by the SAL1, 3’-phosphoadenosine 5’-phosphate (PAP) chloroplast retrograde signalling pathway (Van Aken, & Pogson, 2017).
All plants have two WHY genes (WHY1 and WHY2 ).WHY1 encodes a protein that is located in chloroplasts and nuclei while WHY2 encodes a mitochondria-targeted protein (Desveaux, Maréchal, & Brisson, 2005). WHY1 protein interacts with thylakoid membrane proteins and with the chloroplast nucleoids (Krupinska et al., 2014; Melonek et al., 2010). Unlike many other species, Arabidopsis has a third WHY gene, AtWHY3 that is targeted to plastids (Krause et al., 2005). However, the intracellular localization of the WHY proteins appears to be flexible and determined by developmental and environmental signals. For example, the WHY2 protein that is primarily associated with mitochondrial nucleoids, was found in mitochondria, chloroplasts and nuclei during leaf senescence (Huang et al., 2020). Moreover, it appears that WHY3 can compensate for WHY2 in the Arabidopsis why 2-1  mutant because WHY3 can be targeted to both chloroplasts and mitochondria (Golin et al., 2020). The expression of WHY2 in Arabidopsis decreased the expression of genes encoded by the chondriome (Maréchal et al., 2008). Similarly, the expression of the tomato SlWHY2 in transgenic tobacco plants led to mitochondrial gene transcription and stabilization of mitochondrial functions (Zhao et al., 2018).
Barley leaves deficient in the WHY1 protein have higher levels of chlorophyll than the wild type with an enhanced abundance of plastome-encoded transcripts (Comadira et al., 2015; Krupinska et al. , 2019). In contrast, the leaves of the Arabidopsis why1mutant and why1why3 double mutants are phenotypically similar to the wild type. However, a why1why3polIb-1 triple mutant defective in WHY1, WHY3, and the DNA polymerase 1B (Pol1B) exhibited a severe yellow-variegated phenotype (Lepage, Zampini, & Brisson, 2013). WHY1, WHY3 and RECA1 are associated with the chloroplast RNase H1 AtRNH1C protein and work together to maintain chloroplast genome integrity (Wang et al ., 2021). Maize transposon insertion lines in WHY1 (Zmwhy1-1) have equivalent amounts of chloroplast DNA (cpDNA) to the wild type but are deficient in plastid ribosomes resulting in an albino phenotype (Prikryl, Watkins, Friso, van Wijk, & Barkam, 2008).
We have previously characterized the phenotypes, and metabolite and transcriptome profiles of three RNAi-knockdown barley lines (W1-1, W1-7 and W1-9) that have very low levels of HvWHY1 expression (Comadira et al., 2015). The formation of plastid ribosomes and the establishment of photosynthesis was delayed in the RNAi-knockdown barley lines (Krupinska et al., 2019). Given the growing number of reports showing that WHY1 functions as a transcription factor in the nucleus, regulating the expression of genes involved in a wide range of processes including phytohormone synthesis, development and defence, we posed the hypothesis that WHY1 in the nuclei of developing leaves could also control chloroplast development. We therefore investigated the intracellular distribution of WHY1 between proplastids and nuclei in the bases of developing wild type barley leaves. We also characterized the transcript and metabolite profiles of barley lines (W1-1 and W1-7) lacking WHY1. We discuss the data indicating that the observed delay in plastid development in barley lines lacking WHY1 results from functions of the protein in the nuclei as well as the plastids.